This Multi-PI project combines the efforts of two research groups with different areas of expertise to address the long-term goal of developing bioengineered corneal stroma and endothelial tissues to provide therapy for individuals with corneal blindness. These tissues will be bioengineered from adult stem cells, which can be obtained from the individuals to be treated. Organization of these cells into tissues will be guided by scaffolds constructed of native extracellular matrix proteins, fabricated using a biomimetic, surface-induced assembly process. The Funderburgh Lab at the University of Pittsburgh will obtain stem cells from limbal stroma of donated human corneas. Their extensive work with these corneal stromal stem cells (CSSC) shows that they differentiate to stromal keratocytes and to corneal endothelial cells, tissues responsible for most corneal opacity. Recently we demonstrated that CSSC could be obtained from biopsy samples, presenting the opportunity to generate autologous bioengineered tissues. The Feinberg Lab at Carnegie Melon University has developed a novel approach of assembling native extracellular matrix proteins to produce tissue-like scaffolding with defined 3-D architecture. Aim 1 will focus on assembling bioengineered stroma in which the project will elucidate spatial and biochemical cues provided by the scaffolding and the role of soluble cues (hypoxia, TGF3, FGF2) in generating a stroma-like tissue from CSSC. Individual layers of tissue will be stacked to form multilamellar 3-D tissue similar to that of the corneal stroma. Tissues produced in these experiments will be examined for composition, structure, biomechanical properties, and for the ability to act as a functional substitute for stroma in an in vivo rabbit model. Aim 2 will bioengineer corneal endothelium, based on our observation that CSSC cultured as spheres and then transferred to substratum containing basement membrane proteins form polygonal monolayers and express genes typical of corneal endothelium. Using surface-initiated assembly, the Feinberg group has developed a bioengineered equivalent of Descemet's membrane that increases differentiation, expansion, and function of cultured corneal endothelial cells. Corneal endothelial layers will be generated on these membranes from CSSC, and tested as to gene expression patterns, cell density, cell morphology, and endothelial pump function. Functionality of the constructs will be demonstrated in long-term rabbit models in vivo. This project will use innovative experimental approaches to develop a new understanding of how the insoluble extracellular microenvironment guides formation and function of corneal tissues. Importantly, tissues developed during the study hold the potential of being advanced into clinically relevant studies to provide a novel therapeutic approach to the age-old problem of corneal blindness.